Procaspase 8-mediated disease targeting

ABSTRACT

The invention provides compositions and method for delivering a therapeutic or diagnostic agent to a disease site in a mammal, the method comprising administering to the mammal a therapeutically or diagnostically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises the therapeutic or diagnostic agent coupled to a Procaspase 8 polypeptide and a pharmaceutically acceptable carrier.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a Continuation application of co-pending applicationSer. No. 12/530,504, filed Jul. 10, 2010, now U.S. Pat. No. 8,778,302;which is a National Stage Application of International ApplicationNumber PCT/CA2008/000460, filed Mar. 7, 2008; which claims the benefitof U.S. Provisional Application Ser. No. 60/905,807, filed Mar. 9, 2007and U.S. Ser. No. 60/960,305, filed Sep. 25, 2007, all of which areincorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled “June2012-ST25.txt”which was created on Jun. 8, 2012 and is 52 KB. The entire content ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Secreted Protein, Acidic, Rich in Cysteines (SPARC), also known asosteonectin, is a 281 amino acid glycoprotein which is expressed in thehuman body.

The expression of SPARC is developmentally regulated, with SPARC beingpredominantly expressed in tissues undergoing remodeling during normaldevelopment or in response to injury. See, e.g., Lane et al., FASEB J.,8, 163-173 (1994). For example, high levels of SPARC protein areexpressed in developing bones and teeth, principally osteoblasts,odontoblasts, perichondrial fibroblasts, and differentiatingchondrocytes in murine, bovine, and human embryos. SPARC also playsimportant roles in cell-matrix interactions during tissue remodeling,wound repair, morphogenesis, cellular differentiation, cell migration,and angiogenesis, including where these are associated with diseasestates. For example, SPARC is expressed in renal interstitial fibrosis,and plays a role in the host response to pulmonary insults, such asbleomycin-induce pulmonary fibrosis.

SPARC also is upregulated in several aggressive cancers, but is absentfrom the vast majority of normal tissues. See, e.g., Porter et al., J.Histochem. Cytochem., 43, 791 (1995) and other references identifiedbelow. Indeed, SPARC expression is induced among a variety of tumors(e.g., bladder, liver, ovary, kidney, gut and breast). For example, inbladder cancer, SPARC expression has been associated with advancedcarcinoma, with invasive bladder tumors of stage T2 or greater beingshown to express higher levels of SPARC relative to bladder tumors ofstage T1 (or less superficial tumors). See, e.g., Yamanaka et al., J.Urology, 166, 2495-2499 (2001). In meningiomas, SPARC expression hasbeen associated with invasive tumors only. See, e.g., Rempel et al.,Clincal Cancer Res., 5, 237-241 (1999). SPARC expression also has beendetected in 74.5% of in situ invasive breast carcinoma lesions (see,e.g., Bellahcene et al., Am. J. Pathol., 146, 95-100 (1995)), and 54.2%of infiltrating ductal carcinoma of the breast. See, e.g., Kim et al.,J. Korean Med. Sci., 13, 652-657 (1998). SPARC expression also has beenassociated with frequent microcalcification in breast cancer. See, e.g.,Bellahcene et al., supra (suggesting that SPARC expression may beresponsible for the affinity of breast metastases for the bone).

While SPARC possesses a number of properties, one that has beenexploited is its ability to bind albumin. See, e.g., Schnitzer, J. Biol.Chem., 269, 6072 (1994). One example of the use of this property is in aFDA-approved solvent-free formulation of paclitaxel indicated in thetreatment of metastatic breast cancer, Abraxane® (Abraxis BioScience,Inc., Santa Monica, Calif.). Nab-Paclitaxel utilizes the naturalproperties of albumin to reversibly bind paclitaxel, transport it acrossthe endothelial cell, and concentrate it in areas of tumor. Morespecifically, the mechanism of drug delivery involves, in part,glycoprotein 60-mediated endothelial cell transcytosis ofpaclitaxel-bound albumin and accumulation in the area of tumor byalbumin binding to SPARC. Clinical studies have shown thatnab-paclitaxel is significantly more effective than other paclitaxelformulations, almost doubling the response rate, increasing time todisease progression and increasing survival in second-line patients. SeeGradishar, Expert Opin. Pharmacother. 7(8):1041-53 (2006).

SPARC has affinity for a wide variety of ligands other than albumin,including cations (e.g., Ca2+, Cu2+, Fe2+), growth factors (e.g.,platelet derived growth factor (PDGF), and vascular endothelial growthfactor (VEGF)), extracellular matrix (ECM) proteins (e.g., collagen I-Vand collagen IX, vitronectin, and thrombospondin-1), endothelial cells,platelets, and hydroxyapaptite. As disclosed herein, SPARC alsointeracts with Procaspase 8.

A cascade of protease reactions is responsible for the apoptotic changesobserved in mammalian cells undergoing programmed cell death orapoptosis. This cascade involves members of the aspartate-specificcysteine proteases of the ICE/CED3 family, also known as the Caspasefamily. A variety of stimuli can trigger apoptosis and two majorapoptotic signaling pathways, “extrinsic” and “intrinsic”, convergebiochemically leading to its execution (FIG. 1A). The extrinsic pathwayis triggered by the activation of death receptors, such as Fas; thetumor necrosis factor-related apoptosis-inducing ligand (TRAIL)receptors, DR4 or DR5; or tumor necrosis factor receptor, which triggerdeath signals when bound by their natural ligands. Ligand binding to thereceptor recruits adaptor proteins, such as Fas-associated death domain(FADD), which recruits Procaspase 8 to form death inducing signalingcomplexes (DISCs).

Caspase 8 is activated at DISCs (i.e., converted from Procaspase 8 toCaspase 8 by peptide cleavage), leading to downstream pro-apoptoticevents. The intrinsic pathway is centered around the mitochondria whichis key in regulating the balance between pro- and anti-apoptoticfactors, such as anti-apoptotic members Bcl-2, Bcl-XL and pro-apoptoticmembers Bax, Bak and Bok. It can be triggered by a number of stimuli,including agents that cause DNA damage or growth factor deprivation.This leads to the permeabilization of the mitochondrial membrane, therelease of cytochrome c into the cytosol, which then interacts withAPAF-1 to recruit Caspase 9, resulting in cleavage of executionerCaspases and apoptosis. The convergence of the extrinsic and intrinsicpathways occur when Caspase 8 activates Bid, a Bcl-2 family member thatcan trigger downstream targets to initiate the intrinsic apoptoticpathway.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a composition that directsa therapeutic or diagnostic agent to a disease site in a mammal. Thecomposition comprises a therapeutic or diagnostic agent coupled to aProcaspase 8 polypeptide and a pharmaceutically acceptable carrier. Inparticular, the invention provides a composition comprising atherapeutic or diagnostic agent coupled to a Procaspase 8 polypeptidewith an amino acid sequence comprising one or more of SEQ ID NOS: 1-18,25, 26 and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method fordelivering a therapeutic or diagnostic agent to a disease site in amammal. The method comprises administering to the mammal atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to aProcaspase 8 polypeptide and a pharmaceutically acceptable carrier.

Further, the present invention provides kits for treating or diagnosinga disease in a mammal, the kits comprising a therapeutic or diagnosticagent coupled to a Procaspase 8 polypeptide and instructions for use.

In another aspect, the invention provide pharmaceutical compositions forthe treatment or diagnosis of a disease comprising a therapeutic ordiagnostic agent coupled to a polypeptide comprising the sequence of SEQID NO: 20 or a fragment thereof and a pharmaceutically acceptablecarrier, wherein the polypeptide comprising the sequence of SEQ ID NO:20 or fragment binds to Procaspase 8.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B: Schematic of signaling events in apoptosis: A potentialmodel of SPARC-mediated induction of apoptosis.

FIGS. 2A-2E: The presence of higher levels of SPARC is associated withgreater expression of genes involved in the extrinsic pathway ofapoptosis following exposure to 5-FU.

FIGS. 3A-3D: Inhibition of Caspase 8 Increases Cell Viability in CRCcells with greater SPARC expression.

FIGS. 4A-4C: Inhibition of Caspase 8 gene expression with siRNA enhancessurvival of cells expressing higher levels of SPARC following exposureto 5-FU.

FIGS. 5A-5B: Interaction between Procaspase 8 and SPARC is detected atthe cell membrane.

FIGS. 6A-6D: Caspase 8 is up-regulated in response to over-expression ofSPARC in vitro and in tumor xenografts following treatment with acombination of SPARC and 5-FU in vivo.

FIGS. 7A-7E: Interaction between Procaspase 8 and SPARC is disrupted byan antibody to the Procaspase 8 amino terminal domain.

FIGS. 8A-8B: Interaction between Procaspase 8 and SPARC occurs within aspecific SPARC domain.

FIGS. 9A-9C: Interaction between Bcl-2 and Procaspase 8 occurs in thesame region as where SPARC interacts with Procaspase 8.

FIGS. 10A-10B: Interaction between Bcl-2 and Procaspase 8 occurs in theregion of the DEDI and DEDII domains of Procaspase 8.

DETAILED DESCRIPTION OF THE INVENTION

The apoptotic pathway includes the extrinsic or intrinsic pathways ofapoptosis. The extrinsic pathway involves death receptors (DR4, DR5,brown), adaptor proteins (FADD), and Caspase 8 or 10, while theintrinsic pathway is centered around the mitochondria and involvesCaspase 9 and cytochrome c (FIG. 1A). Without desiring to be bound byany theory, the present invention appears to be based on a novelinteraction (*) between SPARC and Procaspase 8 that results in cleavageof Procaspase 8 (FIG. 1B) following exposure to chemotherapy (5-FU).This leads to the activation of the mitochondrial pathway of apoptosisvia Bid, and appears to be independent of the death receptor activation.Procaspase 8 is known to exist in multiple isoform polypeptides,including mutant forms (see SEQ ID NOS: 1-18, 25, 26).

One aspect of the present invention provides a method for delivering atherapeutic or diagnostic agent to a disease site in a mammal. Themethod comprises administering to the mammal a therapeutically ordiagnostically effective amount of a pharmaceutical compositioncomprising the therapeutic or diagnostic agent coupled to a Procaspase 8polypeptide and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a pharmaceutical compositionfor the treatment or diagnosis of a disease comprising a therapeutic ordiagnostic agent coupled to a polypeptide comprising the sequence of SEQID NO: 20 and a pharmaceutically acceptable carrier, wherein thepolypeptide comprising the sequence of SEQ ID NO: 20 binds to Procaspase8. By “binds to Procaspase 8” it is meant, interacts specifically andwith an adequate avidity to co-immunoprecipitate with a full-length,amino-terminal histidine tagged Procaspase 8 isoform.

Further, the invention provides pharmaceutical compositions for thetreatment or diagnosis of a disease comprising the therapeutic ordiagnostic agent coupled to a polypeptide comprising the fragments ofthe sequence of SEQ ID NO: 20 and a pharmaceutically acceptable carrier,wherein the fragments bind to Procaspase 8. Said fragments are made upof consecutive amino acids in the sequence SEQ ID NO: 20 of a length ofat least 6 amino acids, preferably at least 9 amino acids, morepreferably at least 12 amino acids, even more preferably at least 18amino acids, and most preferably at least 36 amino acids.

Accordingly, the invention also provides a method for delivering atherapeutic or diagnostic agent to a disease site in a mammal comprisingadministering to the mammal a therapeutically or diagnosticallyeffective amount of a pharmaceutical composition comprising thetherapeutic or diagnostic agent coupled to a polypeptide comprising thesequence of SEQ ID NO: 20 or a fragment thereof, and a pharmaceuticallyacceptable carrier, wherein the polypeptide comprising the sequence ofSEQ ID NO: 20 or fragment binds to Procaspase 8.

In the various aspects of the present invention described herein, theProcaspase 8 polypeptide is a polypeptide having at least 70%, desirablyat least 80%, more desirably at least 90% and preferably at least 95%,sequence identity to at least 12 consecutive amino acids (and,desirably, such identity to at least 15, 20, 25, 30, 40, 50, 70, 100,150, 200, 250, 275 or 300 consecutive amino acids) selected from any ofSEQ ID NOS: 1-18, 25, and 26. Most preferably, the Procaspase 8polypeptide has at least 70%, desirably at least 80%, more desirably atleast 90% and preferably at least 95%, sequence identity to any of SEQID NOS: 1-4.

As used herein, “sequence identity” or “identity” in the context of thepolypeptides refers to the residues in the two sequences that are thesame when aligned for maximum correspondence over a specified comparisonwindow. When percentage of sequence identity is used in reference topolypeptides, it is recognized that residue positions which are notidentical often differ by conservative amino acid substitutions, whereamino acid residues are substituted for other amino acid residues withsimilar chemical properties (e.g., charge or hydrophobicity) andtherefore do not change the functional properties of the molecule. Whensequences differ in conservative substitutions, the percent sequenceidentity may be adjusted upwards to correct for the conservative natureof the substitution. Sequences that differ by such conservativesubstitutions are said to have “sequence similarity” or “similarity”.Means for making this adjustment are well known to those of skill in theart. Typically this involves scoring a conservative substitution as apartial rather than a full mismatch, thereby increasing the percentagesequence identity. Thus, for example, where an identical amino acid isgiven a score of 1 and a non-conservative substitution is given a scoreof zero, a conservative substitution is given a score between zeroand 1. The scoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polypeptide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percentage of sequenceidentity.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443 453. An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

In order to further exemplify what is meant by conservative substitutionin the context of the present invention, Groups A-F are listed below.The replacement of one member of the following groups by another memberof the same group is considered to be a conservative substitution.

Group A includes leucine, isoleucine, valine, methionine, phenylalanine,serine, cysteine, threonine, and modified amino acids having thefollowing side chains: ethyl, iso-butyl, —CH₂CH₂OH, —CH₂CH₂CH₂OH,—CH₂CHOHCH₃ and CH₂SCH₃.

Group B includes glycine, alanine, valine, serine, cysteine, threonine,and a modified amino acid having an ethyl side chain.

Group C includes phenylalanine, phenylglycine, tyrosine, tryptophan,cyclohexylmethyl, and modified amino residues having substituted benzylor phenyl side chains.

Group D includes glutamic acid, aspartic acid, a substituted orunsubstituted aliphatic, aromatic or benzylic ester of glutamic oraspartic acid (e.g., methyl, ethyl, n-propyl, iso-propyl, cyclohexyl,benzyl, or substituted benzyl), glutamine, asparagine, CO—NH-alkylatedglutamine or asparagine (e.g., methyl, ethyl, n-propyl, and iso-propyl),and modified amino acids having the side chain —(CH₂)₃COOH, an esterthereof (substituted or unsubstituted aliphatic, aromatic, or benzylicester), an amide thereof, and a substituted or unsubstituted N-alkylatedamide thereof.

Group E includes histidine, lysine, arginine, N-nitroarginine,p-cycloarginine, g-hydroxyarginine, N-amidinocitruline, 2-aminoguanidinobutanoic acid, homologs of lysine, homologs of arginine, andornithine.

Group F includes serine, threonine, cysteine, and modified amino acidshaving C₁-C₅ straight or branched alkyl side chains substituted with —OHor —SH.

The Procaspase 8 polypeptides, SEQ ID NO: 20 polypeptides, and fragmentof SEQ ID NO: 20 contemplated by the present invention may besynthesized, detected, quantified and purified using known technologies.For example, cells expressing exogenous Procaspase 8 polypeptides can begenerated by placing the Procaspase 8 structural gene/cDNA under thecontrol of strong promoter/translation start and the vector transfectedinto mammalian cells to drive the expression of Procaspase 8polypeptides in these cells. Alternatively, Procaspase 8 polypeptidesmay be expressed using bacculovirus or other viruses such as adenovirus.Accordingly, the invention provides for isolated recombinantpolynucleotides encoding polypeptides comprising the amino acidsequences SEQ ID NOS: 1-26 and cells comprising said recombinantpolynucleotides.

Suitable methods of protein detection and quantification include Westernblot, enzyme-linked immunosorbent assay (ELISA), silver staining, theBCA assay (see, e.g., Smith et al., Anal. Biochem., 150, 76-85 (1985)),the Lowry protein assay (described in, e.g., Lowry et al., J. Biol.Chem., 193, 265-275 (1951)) which is a colorimetric assay based onprotein-copper complexes, and the Bradford protein assay (described in,e.g., Bradford et al., Anal. Biochem., 72, 248 (1976)) which dependsupon the change in absorbance in Coomassie Blue G-250 upon proteinbinding. Once expressed, the Procaspase 8 polypeptides may be purifiedby traditional purification methods such as ionic exchange, sizeexclusion, or C18 chromatography.

Procaspase 8 polypeptides, SEQ ID NO: 20 polypeptides, and fragment ofSEQ ID NO: 20 contemplated by the invention can also be prepared bysolid phase synthesis. As is generally known, polypeptides of therequisite length can be prepared using commercially available equipmentand reagents following the manufacturers' instructions for blockinginterfering groups, protecting the amino acid to be reacted, coupling,deprotection, and capping of unreacted residues. Suitable equipment canbe obtained, for example, from Applied BioSystems, Foster City, Calif.,or Biosearch Corporation in San Raphael, Calif. The use of solid phasesynthetic methods is needed if nonencoded amino acids or D-forms ofamino acids are used in the polypeptides. However, for polypeptideswhich are completely made up of amino acids that have codons, one canuse recombinant techniques, e.g, use synthesized DNA sequences incommercially available expression systems.

In another aspect, the present invention provides a method fordelivering a therapeutic or diagnostic agent to a disease site in amammal. This method comprises administering to the mammal atherapeutically or diagnostically effective amount of a pharmaceuticalcomposition comprising the therapeutic or diagnostic agent coupled to anantibody to the Procaspase 8 polypeptide and a pharmaceuticallyacceptable carrier.

It is desirable that the Procaspase 8 polypeptides used in the variousaspects of the present invention are conjugated to polyethylene glycol(PEG). PEG conjugation can increase the circulating half-life of thesepolypeptides, reduce the polypeptide's immunogenicity and antigenicity,and improve their bioactivity. If used, any suitable method of PEGconjugation can be used, including but not limited to, reactingmethoxy-PEG with a Procaspase 8 polypeptide protein's available aminogroup(s) or other reactive sites such as, e.g., histidines or cysteines.In addition, recombinant DNA approaches may be used to add amino acidswith PEG-reactive groups to the Procaspase 8 polypeptides andantibodies. Further, releasable and hybrid PEG-ylation strategies may beused in accordance with the aspects of the present invention, such asthe PEG-ylation of Procaspase 8polypeptide, wherein the PEG moleculesadded to certain sites in the Procaspase 8polypeptide molecule arereleased in vivo. Examples of PEG conjugation methods are known in theart. See, e.g., Greenwald et al., Adv. Drug Delivery Rev. 55:217-250(2003).

The present invention further contemplates that the Procaspase 8polypeptides may include fusion proteins. For example, and withoutlimitation, Procaspase 8 polypeptide sequences may be fused upstream ordownstream of diagnostically useful protein domains (such as hapten,GFP), active protein domains (e.g., without limitation, tTF, TNF, Smar1derived p44 peptide, interferon, TRAIL, Smac, VHL, Procaspase, Caspase,and IL-2) or toxin (e.g., without limitation, ricin, PAP, Diphtheriatoxin, Pseudomonas exotoxin)

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving at least two portions covalently linked together, where each ofthe portions is a polypeptide having a different property. The propertymay be a biological property, such as activity in vitro or in vivo. Theproperty may also be a simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, and the like. Theportions may be linked directly by a single peptide bond or through apeptide linker containing one or more amino acid residues. Generally,the portions and the linker will be in reading frame with each other.

The various aspects of the present invention also contemplate that theProcaspase 8 polypeptide is coupled to a therapeutic or a diagnosticagent. By way of illustration, the coupled moiety may be Procaspase 8polypeptide-radioinuclide, Procaspase 8 polypeptide-drug, Procaspase 8polypeptides-immunomodulator, Procaspase 8 polypeptides-antibody (orantibody fragment) or Procaspase 8 polypeptide-toxin conjugates.

Methods for providing this coupling, e.g., covalent bonding orconjugation, are known to those skilled in the art. For example, andwithout limitation, free amino groups in Procaspase 8 polypeptideproteins or SEQ ID NO: 20 or fragment of SEQ ID NO: 20, such as theepsilon-amino group of lysine, may be conjugated with reagents such ascarodiimides or heterobiofunctional agents. Alternatively, Procaspase 8polypeptide or SEQ ID NO: 20 or fragment of SEQ ID NO: 20 groups can beused for conjugation. Sugar moieties bound to Procaspase 8 polypeptideglycoproteins also may be oxidized to form aldehydes groups useful in anumber of coupling procedures known in the art. The conjugates formed inaccordance with the invention can be stable in vivo or labile, such asenzymatically degradeable tetrapeptide linkages or acid-labilecis-aconityl or hydrazone linkages.

Numerous heterobifunctional cross-linking reagents that are used to formcovalent bonds between amino groups and thiol groups and to introducethiol groups into proteins, are known to those of skill in this art(see, e.g., Cumber et al. (1992) Bioconjugate Chem. 3:397-401; Thorpe etal. (1987) Cancer Res. 47:5924-5931; Gordon et al. (1987) Proc. Natl.Acad. Sci. 84:308-312; Walden et al. (1986) J. Mol. Cell Immunol.2:191-197; Carlsson et al. (1978) Biochem. J. 173: 723-737; Mahan et al.(1987) Anal. Biochem. 162:163-170; Wawryznaczak et al. (1992) Br. J.Cancer 66:361-366; Fattom et al. (1992) Infection & Immun. 60:584-589).These reagents may be used to form covalent bonds between a Procaspase 8polypeptide and any of the active agents disclosed herein. Thesereagents include, but are not limited to:N-succinimidyl-3-(2-pyridyidithio)propionate (SPDP; disulfide linker);sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate(sulfo-LC-SPDP); succinimidyloxycarbonyl-.alpha.-methyl benzylthiosulfate (SMBT, hindered disulfate linker); succinimidyl6-[3-(2-pyridyidithio)propionamido]hexanoate (LC-SPDP);sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-SMCC); succinimidyl 3-(2-pyridyldithio)butyrate (SPDB; hindereddisulfide bond linker); sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3-dithiopropionate(SAED); sulfo-succinimidyl 7-azido-4-methylcoumarin-3-acetate (SAMCA);sulfosuccinimidyl6-[alpha-methyl-alpha-(2-pyridyidithio)toluamido]hexanoate(sulfo-LC-SMPT); 1,4-di-[3′-(2′-pyridyidithio)propionamido]butane(DPDPB);4-succinimidyloxycarbonyl-.alpha.-methyl-.alpha.-(2-pyridylthio)-toluene(SMPT, hindered disulfate linker);sulfosuccinimidyl6[.alpha.-methyl-.alpha.-(2-pyridyldithio)toluamido]hexa-noate(sulfo-LC-SMPT); m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB; thioether linker);sulfosuccinimidyl(4-iodoacetyl)amino benzoate (sulfo-SIAB);succinimidyl4(p-maleimidophenyl)butyrate (SMPB);sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB);azidobenzoyl hydrazide (ABH).

Other heterobifunctional cleavable coupling agents include,N-succinimidyl (4-iodoacetyl)-aminobenzoate; sulfosuccinimydil(4-iodoacetyl)-aminobenzoate;4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)-toluene;sulfosuccinimidyl-6-[a-methyl-a-(pyridyldithiol)-toluamido]hexanoate;N-succinimidyl-3-(-2-pyridyidithio)-proprionate; succinimidyl6[3(−(-2-pyridyldithio)-proprionamido]hexanoate; sulfosuccinimidyl 6[3(−(-2-pyridyldithio)-propionamido]hexanoate;3-(2-pyridyidithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, S-(2-thiopyridyl)-L-cysteine. Further exemplarybifunctional linking compounds are disclosed in U.S. Pat. Nos.5,349,066, 5,618,528, 4,569,789, 4,952,394, and 5,137,877.

The Procaspase 8 polypeptide or SEQ ID NO: 20 or fragment of SEQ ID NO:20 is optionally linked to the active agent via one or more linkers.Flexible linkers and linkers that increase solubility of the conjugatesare contemplated for use, either alone or with other linkers are alsocontemplated herein. The linker moiety is selected depending upon theproperties desired. For example, the length of the linker moiety can bechosen to optimize the kinetics and specificity of ligand binding,including any conformational changes induced by binding of the ligand toa target receptor. The linker moiety should be long enough and flexibleenough to allow the polypeptide ligand moiety and the target cellreceptor to freely interact. If the linker is too short or too stiff,there may be steric hindrance between the Procaspase 8 polypeptidemoiety and the cell toxin. If the linker moiety is too long, the activeagent may be degraded in the process of production, or may not deliverits desired effect to the target cell effectively. In some embodiments,several linkers may be included in order to take advantage of desiredproperties of each linker.

Any suitable linker known to those of skill in the art can be usedherein. Linkers and linkages that are suitable for chemically linkedconjugates include, but are not limited to, disulfide bonds, thioetherbonds, hindered disulfide bonds, and covalent bonds between freereactive groups, such as amine and thiol groups. These bonds areproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the polypeptides and then reacting the thiolgroups on one polypeptide with reactive thiol groups or amine groups towhich reactive maleimido groups or thiol groups can be attached on theother.

Peptide linkers may also be linked by expressing DNA encoding theProcaspase 8 polypeptide or SEQ ID NO: 20 or fragment of SEQ ID NO: 20,linker and, optionally, active agent as a fusion protein. Accordingly,linkers can include, but are not limited to, peptidic linkages, aminoacid and peptide linkages, typically containing between one and about 60amino acids, preferably between about 5 and 30 amino acids, morepreferably between about 10 and 20 amino acids. In addition, theProcaspase 8 polypeptide or SEQ ID NO: 20 polypeptide or fragment of SEQID NO: 20 provided by the invention may have between one and about 60amino acids, preferably between about 5 and 30 amino acids, morepreferably between about 10 and 20 amino acids added to its amino orcarboxyl terminus.

As used herein, the term “therapeutic agent” refers to a chemicalcompound, a biological macromolecule, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues that are suspected of having therapeuticproperties, e.g., chemotherapeutic agent or radiotherapy agent. Theagent may be purified, substantially purified or partially purified.

Illustrative of the therapeutic agents which may be coupled to theProcaspase 8 polypeptide or SEQ ID NO: 20 or fragment of SEQ ID NO: 20,in the manner contemplated by the present invention include, withoutlimitation, chemotherapeutic agents (e.g., docetaxel, paclitaxel,taxanes and platinum compounds), antifolates, antimetabolites,antimitotics, DNA damaging agents, proapoptotics, differentiationinducing agents, antiangiogenic agents, antibiotics, hormones, peptides,antibodies, tyrosine kinase inhibitors, biologically active agents,biological molecules, radionuclides, adriamycin, ansamycin antibiotics,asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine,capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin,dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel,doxorubicin, etoposide, epothilones, floxuridine, fludarabine,fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan,methotrexate, rapamycin (sirolimus), mitomycin, mitotane, mitoxantrone,nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin,procarbazine, rituximab, streptozocin, teniposide, thioguanine,thiotepa, taxanes, vinblastine, vincristine, vinorelbine, taxol,combretastatins, discodermolides, transplatinum, anti-vascularendothelial growth factor compounds (“anti-VEGFs”), anti-epidermalgrowth factor receptor compounds (“anti-EGFRs”), 5-fluorouracil andderivatives, a radionuclide, a kinase inhibitor (e.g., genistein).

As used herein, the term “chemotherapeutic agent” refers to an agentwith activity against cancer, neoplastic, and/or proliferative diseases.Suitable chemotherapeutic agents (which includes compounds referred toas anticancer agents) that may be used accordance with the presentinvention include, but are not limited to, tyrosine kinase inhibitors(genistein), biologically active agents (TNF, of tTF), radionuclides(¹³¹I, ⁹⁰Y, ¹¹¹In, ²¹¹At, ³²P and other known therapeuticradionuclides), adriamycin, ansamycin antibiotics, asparaginase,bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine,chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine,dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin,etoposide, epothilones, floxuridine, fludarabine, fluorouracil,gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin(sirolimus) and derivatives, mitomycin, mitotane, mitoxantrone,nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin,procarbazine, rituximab, streptozocin, teniposide, thioguanine,thiotepa, taxanes, vinblastine, vincristine, vinorelbine, taxol,combretastatins, discodermolides, transplatinum, antimetabolites (e.g.,asparaginase), antimitotics (e.g., vinca alkaloids), DNA damaging agents(e.g., cisplatin), proapoptotics (agents which induceprogrammed-cell-death or apoptosis) (e.g, epipodophylotoxins),differentiation inducing agents (e.g., retinoids), antibiotics (e.g.,bleomycin), hormones (e.g., tamoxifen, diethylstibestrol),antiangiogenesis agents (angiogenesis inhibitors, e.g., INF-alpha,fumagillin, angiostatin, endostatin, thalidomide, and the like),biologically active polypeptides, antibodies, lectins, and toxins.

Preferred chemotherapeutic agents include docetaxel and paclitaxel asparticles comprising albumin wherein more than 50% of thechemotherapeutic agent is in nanoparticle form. Most preferably, thechemotherapeutic agent comprises particles of albumin-bound paclitaxel,e.g., Abraxane®.

The pharmaceutical compositions may also include, if desired, additionaltherapeutic or biologically-active agents. For example, therapeuticfactors useful in the treatment of a particular indication can bepresent. Factors that control inflammation, such as ibuprofen orsteroids, can be part of the composition to reduce swelling andinflammation associated with in vivo administration of thepharmaceutical composition and physiological distress.

The term “therapeutic” as used herein refers to curing or preventing,the latter illustrated by the prevention or lessening the chance of atargeted disease (e. g., cancer or other proliferative disease) orrelated condition thereto afflicting a subject mammal. Curative therapyrefers alleviating, in whole or in part, an existing disease orcondition in a mammal.

The term “therapeutically effective amount” it is meant an amount thatreturns to normal, either partially or completely, physiological orbiochemical parameters associated with or causative of a disease orcondition. A clinician skilled in the art should be able to determinethe amount of the pharmaceutical composition that will betherapeutically effective relative to a particular disease or condition.By way of example, and in accordance with a preferred embodiment whereinthe therapeutic agent is paclitaxel, the paclitaxel dose administeredmay range from about 30 mg/m² to about 1000 mg/m² with a dosing cycle ofabout 3 weeks (i.e., administration of the paclitaxel dose once everyabout three weeks), desirably from about 50 mg/m² to about 800 mg/m²,preferably from about 80 mg/m² to about 700 mg/m², and most preferablyfrom about 250 mg/m² to about 300 mg/m² with a dosing cycle of about 3weeks.

The invention provides embodiments wherein the Procaspase 8 polypeptideor SEQ ID NO: 20 or fragment of SEQ ID NO: 20, is fused or coupled to anantibody or antibody fragment which mediates one or more of complementactivation, cell mediated cytotoxicity, and opsinization. The term“antibody” herein includes, without limitation, monoclonal antibodies,polyclonal antibodies, dimers, multimers, multispecific antibodies(e.g., bispecific antibodies). Antibodies may be murine, human,humanized, chimeric, or derived from other species. An antibody is aprotein generated by the immune system that is capable of recognizingand binding to a specific antigen. A target antigen generally hasnumerous binding sites, also called epitopes, recognized by CDRs onmultiple antibodies. Each antibody that specifically binds to adifferent epitope has a different structure. Thus, one antigen may havemore than one corresponding antibody. An antibody includes a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) orsubclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) of immunoglobulinmolecule. The immunoglobulins can be derived from any species.

“Antibody fragments” comprise a portion of a full length antibody, whichmaintain the desired biological activity. “Antibody fragments” aregenerally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g., Old World Monkey or Ape) and human constant regionsequences.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express Fc.gamma.RIII only, whereas monocytes expressFc.gamma.RI, FcγRII and FcγRIII. To assess ADCC activity of a moleculeof interest, an in vitro ADCC assay may be performed (U.S. Pat. No.55,003,621; U.S. Pat. No. 5,821,337). Useful effector cells for suchassays include peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al PNAS (USA), 95:652-656 (1998).

An antibody which “induces cell death” is one which causes a viable cellto become nonviable. Cell death in vitro may be determined in theabsence of complement and immune effector cells to distinguish celldeath induced by antibody-dependent cell-mediated cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC). Thus, the assay for cell deathmay be performed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the antibody is able to induce cell death, loss of membraneintegrity as evaluated by uptake of propidium iodide (PI), trypan blueor 7AAD can be assessed relative to untreated cells. Cell death-inducingantibodies are those which induce PI uptake in the PI uptake assay inBT474 cells.

An antibody which “induces apoptosis” is one which induces programmedcell death as determined by binding of annexin V, fragmentation of DNA,cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation,and/or formation of membrane vesicles (called apoptotic bodies).

Turning to the diagnostic aspect of the present invention, thediagnostic agents that may be used include, without limitation,radioactive agents, MRI contrast agents, X-ray contrast agents,ultrasound contrast agents, and PET contrast agents. The coupling ofthese agents, described in connection with therapeutic agents, is alsocontemplated by this aspect of the invention. Further, the term“diagnostically effective amount” is an amount of the pharmaceuticalcomposition that in relevant clinical settings allows for a reasonablyaccurate determination of the presence of abnormal proliferative,hyperplastic, remodeling, inflammatory activity in tissues and organs.For example, the condition “diagnosed” in accordance with the inventioncan be a benign or malignant tumor.

For use in vivo, the chemotherapeutic agent coupled Procaspase 8polypeptide or SEQ ID NO: 20 or fragment of SEQ ID NO: 20, is desirablyformulated into a pharmaceutical composition comprising aphysiologically acceptable carrier. Any suitable physiologicallyacceptable carrier can be used within the context of the invention,depending on the route of administration. Those skilled in the art willappreciate those carriers that may be used in to provide apharmaceutical composition suitable for the desired method ofadministration.

The administration of the pharmaceutical compositions of the presentinvention may be accomplished via any suitable route including, but notlimited to, intravenous, intraperitoneal, intratumoral, and inhalationaladministration, with intravenous and intratumoral administration beingmost preferred.

In the case of inhalational therapy, the pharmceutical composition ofthe present invention is desirably in the form of an aerosol. Aerosoland spray generators for administering the agent if in solid form areavailable. These generators provide particles that are respirable orinhalable, and generate a volume of aerosol containing a predeterminedmetered dose of a medicament at a rate suitable for humanadministration. Examples of such aerosol and spray generators includemetered dose inhalers and insufflators known in the art. If in liquidform, the pharmaceutical compositions of the invention may beaerosolized by any suitable device.

When used in connection with intravenous, intraperitoneal orintratumoral administration, the pharmaceutical composition of theinvention may comprise sterile aqueous and non-aqueous injectionsolutions, suspensions or emulsions of the active compound, whichpreparations are preferably isotonic with the blood of the intendedrecipient. These preparations may contain one or more of anti-oxidants,buffers, surfactants, cosolvents, bacteriostats, solutes which renderthe compositions isotonic with the blood of the intended recipient, andother formulation components known in the art. Aqueous and non-aqueoussterile suspensions may include suspending agents and thickening agents.The compositions may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials.

The methods of the present invention may also be part of combinationtherapy. The phrase “combination therapy” refers to administering atherapeutic agent in accordance with the invention together with anothertherapeutic composition in a sequential or concurrent manner such thatthe beneficial effects of this combination are realized in the mammalundergoing therapy.

The methods of the invention are suitable for use in diagnosing ortreating various diseases including, but not limited to, abnormalconditions of proliferation, tissue remodeling, hyperplasia, exaggeratedwound healing in any bodily tissue including soft tissue, connectivetissue, bone, solid organs, blood vessel and the like. More specificexamples of such diseases include cancer, diabetic or other retinopathy,inflammation, fibrosis, arthritis, restenosis in blood vessels orartificial blood vessel grafts or intravascular devices and the like.

In a preferred aspect, the invention provides methods of diagnosingand/or treating a tumor, wherein the tumor is selected from the groupconsisting of oral cavity tumors, pharyngeal tumors, digestive systemtumors, the respiratory system tumors, bone tumors, cartilaginoustumors, bone metastases, sarcomas, skin tumors, melanoma, breast tumors,the genital system tumors, urinary tract tumors, orbital tumors, brainand central nervous system tumors, gliomas, endocrine system tumors,thyroid tumors, esophageal tumors, gastric tumors, small intestinaltumors, colonic tumors, rectal tumors, anal tumors, liver tumors, gallbladder tumors, pancreatic tumors, laryngeal tumors, tumors of the lung,bronchial tumors, non-small cell lung carcinoma, small cell lungcarcinoma, uterine cervical tumors, uterine corpus tumors, ovariantumors, vulvar tumors, vaginal tumors, prostate tumors, prostaticcarcinoma, testicular tumors, tumors of the penis, urinary bladdertumors, tumors of the kidney, tumors of the renal pelvis, tumors of theureter, head and neck tumors, parathyroid cancer, Hodgkin's disease,Non-Hodgkin's lymphoma, multiple myeloma, leukemia, acute lymphocyticleukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronicmyeloid leukemia. In addition, the invention provides for method ofpredicting or determining a tumor's response to a chemotherapeuticagent, methods of treating a tumor, and kits for predicting the responseof a mammalian tumor to a chemotherapeutic agent, wherein the tumor is asarcoma, adenocarcinoma, squamous cell carcinoma, large cell carcinoma,small cell carcinoma, basal cell carcinoma, clear cell carcinoma,oncytoma or combinations thereof.

Further, and in a related aspect, the invention provides a method ofpredicting or determining a tumor's response to a chemotherapeuticagent, as well as a method of predicting or determining a proliferativedisease's response to a chemotherapeutic agent or treating aproliferative disease, including but, not limited to, where theproliferative diseases is, e.g., benign prostatic hyperplasia,endometriosis, endometrial hyperplasia, atherosclerosis, psoriasis or aproliferative renal glomerulopathy.

The invention provides for embodiments wherein the disease is in amammal, including but not limited to, a human.

The following examples further illustrate the invention but should notbe construed as in any way limiting its scope.

Example 1

This example demonstrates the specific binding of anti-SPARC antibody toSPARC.

Whole cell extract was prepared from HUVEC cells by sonication. Theprotein was separated on a 5-15% SDS-PAGE, transferred onto PVDFmembrane and visualized with a polyclonal antibody against SPARC and amonoclonal antibody against SPARC. Both antibodies reacted to a singleband at 38 kDa, the correct molecular weight for SPARC. When MX-1 tumorcell line was analyzed by the same method, SPARC was detected in boththe clarified cell lysate or the membrane rich membrane fraction.

Example 2

This example demonstrates the absence of SPARC expression in normaltissues.

Normal human and mouse tissue were immunostained and scored (0-4) forSPARC staining using a tumor and normal tissue array. Immunostaining wasperformed using polyclonal rabbit anti-SPARC antibody. SPARC was notexpressed in any of the normal tissues, with the exception of theesophagus. Likewise, SPARC was not expressed in any of the normal mousetissue, except the kidney of the female mouse. However, it is possiblethat this expression was due to follistatin which is homologous toSPARC.

SPARC Expression in Human Normal Tissues

Stomach 0/8 Colon 0/9 Rectum 0/15 Liver 0/14 Spleen 0/10 Lung 0/14Kidney 1/14 Brain 1/14 Testis 0/8 Prostate 0/3 Heart 0/9 Tonsil 0/10Lymph Nodes 0/10 Appendix 0/10 Esophagus 5/5 Pancreas 0/5 Eyeball 0/5Ovary 0/5

Mouse Normal Tissues

Liver 0/19 Kidney (M) 0/8 Kidney (F) 6/8 Lung 0/16 Muscle 0/20 Brain0/20 Heart 0/18 Stomach 0/20 Spleen 0/20

Example 3

This example illustrates the expression of SPARC in MX-1 tumor cells.

MX-1 cells were cultured on a coverslip and stained with an antibodydirected against human SPARC using methods known in the art. Antibodystaining was observed, which demonstrates that MX-1 is expressing SPARC.These results suggest that SPARC expression detected in MX-1 tumor cellsis a result of SPARC secretion by MX-1 tumor cells. Staining was moreintense for MX-1 tumor cells than that of normal primary cells such asHUVEC (human umbiblical vein endothelial cells), HLMVEC (Human lungmicrovessel endothelial cells), and HMEC (Human mammary epithelialcells). Though the majority of the SPARC staining was internal SPARC,significant level of surface SPARC was detected as demonstrated byconfocal miscroscopy and staining of unpermeabilized cells.

Example 4

This example illustrates the overexpression of SPARC protein in humanbreast carcinoma cells.

SPARC expression in human breast carcinoma cells was determined using atumor array from Cybrdi, Inc. (Gaithersburg, Md.). The results of thisanalysis are set forth in Table 1. Intensity of staining was scored from“Negative” to 4+, with the higher number corresponding to greaterintensity of overexpression. 49% of breast carcinoma stained positive(2+ and above) for SPARC, as compared to 1% of normal tissue (p<0.0001).

SPARC Staining (%) Negative −/+ 1+ 2+ 3+ 4+ Carcinoma Cells 31 14 1 11 925 (34%) (15%) (1%) (12%) (10%) (27%) Normal Cells 93 7 4 1 0 0 (89%) (7%) (4%)  (1%)  (0%)  (0%)

Example 5

This example demonstrates SPARC overexpression in squamous cell head andneck cancers with high response rates using nanoparticle albumin-boundpaclitaxel (ABI-007).

In phase I and II clinical studies of patients with squamous cellcarcinoma (SCC) of head and neck (H&N) and anal canal, response rates of78% and 64% were observed, respectively, for intra-arterially deliveredNanoparticle Albumin-Bound Paclitaxel (Abraxane®, ABX or ABI-007) (see,e.g., Damascelli et al., Cancer, 92(10), 2592-2602 (2001), andDamascelli et al., AJR, 181, 253-260 (2003)). In comparing in vitrocytoxicity of ABX and Taxol (TAX), we observed that a squamous cervix(A431) line demonstrated improved IC₅₀s for ABX (0.004 μg/ml) vs TAX(0.012 μg/ml). Albumin-mediated transendothelial caveolar transport ofpaclitaxel (P) and increased intratumoral accumulation of P for ABX vsTAX was demonstrated recently (see, e.g., Desai, SABCS 2003).

Human H&N tumor tissues (n=119) and normal human H&N tissue (n=15) wereimmunostained and scored (0-4+) for SPARC staining using a tumor andnormal tissue array. Immunostaining was performed using polyclonalrabbit anti-SPARC antibody. In a new phase I dose escalation study (ABXgiven IV over 30 minutes q3w), a subset of head and neck cancer patients(n=3) were analyzed for response to ABX.

SPARC was overexpressed (score >2+) in 60% (72/119) of the H&N tumorsversus 0% (0/15) in normal tissues (p<0.0001).

SPARC Staining (%) Negative −/+ 1+ 2+ 3+ 4+ H&N Tumor 17 14 16 23 20 29Array: (14%) (12%) (13%) (19%) (17%) (24%) Carcinoma Cells Normal Cells13 0 2 0 0 0 (87%)  (0%) (13%)  (0%)  (0%)  (0%)

In a new phase I dose escalation study (ABX given IV over 30 minutesq3w), a subset of head and neck cancer patients (n=3) were analyzed forresponse to ABX. In this study, 2/3 H&N patients achieved partialresponse (PR) after 2 cycles of treatment at

dose levels of 135 mg/m² (1 pt) and 225 mg/m² (1 pt). A third patient at260 mg/m² progressed. Tumor tissues from these patients were stained forSPARC and 1 of the responding patients showed strong overexpression forSPARC.

Example 6 1. Materials and Methods

Cell Lines: MIP101 and HCT116 (ATCC) human CRC cells were maintained inDMEM media supplemented with 1% penicillin-streptomycin, 1% kanamycin(Invitrogen) and 10% newborn calf serum at 37° C. and 5% CO2. For MIP101cells resistant to 5-FU (MIP/5FU) or CPT-11 (MIP/CPT), media were alsosupplemented with 500 μM 5-FU or 10 μM CPT-11, respectively. MIP101transfected with empty vector (MIP/ZEO) and MIP101 cells stablytransfected with SPARC (MIP/SP), were also supplemented with 0.01%zeocin (Invitrogen).

RT-PCR: Cells were seeded at 150,000 cells/well in 6-well plates. After24 hours, cells were incubated with 1000 μM 5-FU for 0-4 hours and RNAisolated 24 hours later with Trizol (Invitrogen) [12]. 1 μg of total RNAwas used to generate cDNA (Superscript III, Invitrogen). Specificprimers were used as previously described for SPARC [12]; othersinclude:

caspase 8: 5′-ATCACAGACTTTGGACAAAGTTTA-3′ (sense),5′-TCTGAATCAGTCTCAACAGGTATA-3′ (anti-sense); caspase 10:5′-AAGCTTCTGATTATTGATTCAAACC-3′ (sense), 5′-TTCTCTATGTTTCTCAAAAGTTTA- 3′(anti-sense); DR4:  5′-GGACAATGCTCACAACGAGA-3′ (sense), 5′-TGTTGACCCATTTCATCAGC-3′ (anti-sense); FADD: 5′-TGTGCAGCATTTAACGTCATATGT-3′ (sense),  5′-ACGCAGCTTGAGTTCAGAA-3′(antisense); TRADD:  5′-TTTGAGTTGCATCCTAGCCCA-3′ (sense), 5′-GCTGGTGAGCTCGTTCTC-3′ (anti-sense); and β-actin: 5′-GCCACGGCTGCTTCCAG-3′ (sense), 5′-GGCGTACAGGTCTTTC-3′ (anti-sense).

PCR settings: 94° C. for 3-5 minutes, followed by 32-41 cycles at: 94°C. for 20-60 seconds; followed by: (SPARC): 65° C. for 1 minute,(caspase 8, DR4 and β-actin): 58° C. for 15 seconds, (caspase 10 andTRADD): 50° C. for 15 seconds, (FADD): 55° C. for 15 seconds; then 72°C. for 30-60 seconds; followed by extension at 72° C. for 7-10 minutes.PCR products were separated on a 1.5% agarose gel electrophoresis,imaged used for quantitation of expression levels, and values normalizedto β-actin levels.

Immunoblot Analysis: 48 hours after seeding, cells were incubated with1000 μM 5FU, and collected at 0-12 hours for protein. 40 μg totalprotein/sample was loaded, separated on a 12% SDS-PAGE, then transferredto PDVF membranes (Bio-Rad). Immunodetection was performed usingantibodies against Caspase 8, FADD, p-FADD, Caspase 10, BID, Caspase 9,and Caspase 3 (all 1:1000, Cell Signaling Technologies); and cleavedCaspase 8 (1:1000, Calbiochem), followed by incubation with theappropriate secondary antibody. All immunoblots were also probed withantibodies to β-actin (0.32 μg/mL, Abeam) as loading control. Proteinswere detected with SuperSignal West Dura (Pierce).

RNA Interference: Initially, to assess the efficiency of Caspase 8 geneexpression knock-down by siRNA, MIP/SP and HCT116 cells were seeded(6-well plate). 24 hours later, cells were transiently transfected with20-60 nM scramble oligonucleotide sequence (control), or Caspase 8 siRNA(Stealth RNAi, Invitrogen) and cells collected at various time intervalsfollowing transfection. 40 nM of siRNA yielded the most efficientknock-down (14-fold decrease in Caspase 8 expression at 48-96 hours).For all subsequent experiments, 40 nM of siRNA or scramble control wasused. Following Caspase 8 siRNA transfection, cells were assessed forcell viability, and apoptosis using either Caspase 3/7 assay or TUNELassay.

Cell Viability Assay: 24 hours after seeding (˜60% confluence), cellswere transiently transfected with Caspase 8 siRNA for 48 hours beforeincubation with 1000 μM 5-FU or 100 μM CPT-11 for 48 hours. Cellviability was assessed by MTS assay (Promega) at 490 nm. Caspase 3/7Assay: Cells were transiently transfected with 40 nM of Caspase 8 siRNAfor 48 hours, and incubated with 1000 μM 5-FU for another 48 hours.Total cell lysates were isolated and 20 μg of total protein/sample wereused in Caspase-Glo 3/7 Assay (Promega), using a 1:1 dilution ofCaspase-Glo 3/7 Substrate. Relative luminescence units (RLU) wasquantified using a Viktor2±1420 Multilabel counter (Perkin Elmer).

TUNEL Assay: Cells were seeded (24-well plates) to achieve ˜60%confluence 24 hours later for transient transfection with Caspase 8siRNA. 36 hours later, cells were incubated with 1000 μM 5-FU for 36hours, harvested (suspension and attached cells) and fixed onto glassslides with Shandon cytospin at 2000 rpm for 10 minutes and stained asper manufacturer's instructions (Promega). The number of TUNEL-positivecells was counted and averaged from four different fields (n=4independent experiments, with slides read independently by twoindividuals in a blinded fashion).

caspase 8/9 Inhibition: Cells were seeded (96-well plates), andincubated 24 hours later (˜60% confluence) with 10-50 μM of Caspase8-like inhibitor (z-IETD-fmk, Sigma) or Caspase 9-like inhibitor(z-LEHDfmk•TFA, Sigma) for 30 minutes, followed by incubation with 1000μM 5-FU for an additional 24 hours. Cell viability was assessed by MTSassay.

Subcellular Fractionation and Immunoprecipitation: MIP/SP and HCT116cells were grown until ˜80% confluence, incubated with 1000 μM 5-FU andisolated at 0-4 hours. Cells were separated into nuclear, cytosolic, andmembrane fractions using ProteoExtract Subcellular Proteome ExtractionKit (EMD Biosciences Inc.). 250 μg of the individual cellular fractionswere incubated with α-SPARC (10 μg/mL, Haematologic Technologies),α-caspase 8 (1:100) or a non-specific anti-mouse IgG antibody as control(Cell Signaling Technologies), in PBS overnight (4° C.) with gentleagitation. Protein:Antibody mixture was then incubated with 30 μL ofProtein A: Protein G (Sigma) (1:1) beads for 4 hours (4° C.). Proteinswere also incubated with EZView Red His-Select HC Nickel affinity gel(Sigma) for immunoprecipitation of His-tagged SPARC protein. For allcomplexes, beads were washed 5× with PBS, eluted with 40 μL of2×SDS-Loading Buffer, and used for immunoblotting against SPARC, Caspase8, p-FADD, and DR4 (0.5 μg/mL, Santa Cruz).

Animal Studies: Tumors harvested from xenografts from animals (NIH nudemice, 6 weeks old, Taconic Laboratories) were used for histology, RT-PCRor immunoblot. 2×106 MIP101 cells were injected into the left flank.Once tumors reached 100 mm³, animals were treated with chemotherapyusing 3-week cycle regimen (×2 cycles) as previously described (Tai IT,Dai M, Owen D A, Chen L B: Genome-wide expression analysis oftherapy-resistant tumors reveals SPARC as a novel target for cancertherapy. J Clin Invest 2005; 115: 1492-502.)

Experimental groups (2 animals/group) for this study included treatmentwith: SPARC, SPARC+5-FU, 5-FU only, and saline. In addition, tumorxenografts of MIP/ZEO and MIP/SP cells from nude mice treated witheither 5-FU (three consecutive days) or saline, were collected after the1st cycle of treatment and homogenized (Kinematica, POLYTRON-Aggregate).Lysates were then prepared for immunoblot or RT-PCR. All animalsreceived care according to standard animal care protocol and guidelines.For histology, tissue sections of tumor xenografts were processed forimmunohistochemistry based on previously established protocols (Tai I T,Dai M, Owen D A, Chen L B: Genome-wide expression analysis oftherapy-resistant tumors reveals SPARC as a novel target for cancertherapy. J Clin Invest 2005; 115: 1492-502.). Similarly, cells seeded oncoverslips at 150,000 cells/well in a 6-well plate, treated 24 hourslater with 1000 μM 5-FU for 2 hours were then fixed in 2%paraformaldehyde and processed for immunofluorescence staining aspreviously described [27]. In both cases, α-caspase 8 (1:50,paraffin-embedded tissues; 1:100, cells on coverslips) antibody was usedand incubated overnight at 4° C., and counterstained with DAPI. ZeissAxioplan 2 Fluorescence microscope was used for image capture.

Statistics: Statistical difference between experimental groups werecalculated and analyzed using Student's t-test. Statistical significancewas defined as p<0.05, using Smith's Statistical Package.

2. Results

SPARC-over expressing MIP101 cells have higher levels of expression ofgenes involved in the extrinsic pathway of apoptosis.

Over-expression of SPARC in MIP 101 CRC cells (MIP/SP) leads toincreased sensitivity to 5-FU and CPT-11 chemotherapy by diminishingcell survival and enhancing apoptosis. In order to understand themechanisms involved in this SPARC-mediated effect, studies began byexamining the relative contribution of genes involved in apoptosis atthe transcriptional level. Total RNA isolated from MIP/SP andempty-vector control cells (MIP/ZEO) were used to determine theexpression levels of those involved in the extrinsic pathway ofapoptosis, such as Caspase 8, Caspase 10, DR4, and FADD. It was notedthat Caspase 8, 10 and FADD appeared significantly higher in MIP/SPcells, while DR4 expression was greater in control MIP/ZEO cells (FIG.2A). The greatest difference in gene expression was observed withCaspase 8 and 10, which were 6.7 and 4.8-fold higher in MIP/SP thanMIP/ZEO respectively, thereby suggesting that differential expression ofSPARC positively influenced genes involved in the extrinsic pathway ofapoptosis. SPARC over-expression leads to greater activation of theextrinsic pathway of apoptosis

The results noted above show a correlation between SPARC and Caspase 8and 10 levels in MIP101 cells. This prompted an assessment of whetherthe extrinsic pathway may be involved in SPARC-mediated apoptosis. Ithad been observed a greater number of cells undergoing apoptosis wheneither sensitive MIP101 or 5-FU resistant MIP101 cells (MIP/5FU) wereexposed to SPARC in combination with 5-FU in vitro and in vivo. Based onthese findings, the effect of 5-FU exposure on the extrinsic pathway incells with variable levels of SPARC was studied (highest in MIP/SP,moderate in MIP/ZEO and lowest in MIP/5FU). Higher levels of Caspase 8and 10 gene expression were observed in MIP/SP cells and this increasedfollowing exposure to 5-FU 1000 μM. In cells with low SPARC expression,Caspase 8 and 10 were not observed either basally or following exposureto 5-FU in both MIP/ZEO or MIP/5FU cells (FIG. 2B). FADD gene expressionincreased in all cells following treatment with 5-FU, while nosignificant change was noted with DR4 expression with exposure to 5-FUin MIP/SP or MIP/5FU while a decrease was noted in MIP/ZEO. Thisheightened basal expression of Caspase 8 and 10 at the transcriptionallevel in cells over-expressing SPARC translated to the protein level,where MIP/SP cells again showed an abundance of pro-caspase 8 and 10, incomparison to MIP/ZEO and MIP/5FU cells prior to exposure to 5-FU (FIG.2C). Conversion from the 57 kDa pro-caspase 8 to a cleaved product of 48kDa occurred following treatment with 1000 μM of 5-FU. There were alsohigher basal levels of Bid in MIP/SP cells, which peaked at 4 hoursafter treatment with 5-FU followed by a gradual decline over the next 8hours. Interestingly, following 5-FU treatment, activation of Caspases 9and 3 was prominently observed in MIP/SP cells and to a lesser degree inMIP/ZEO, and even less in MIP/5FU cells. FADD was basally expressed inall cell lines, however, significantly greater phosphorylated FADD wasseen in MIP/SP as early as 4 hours after treatment with 5FU incomparison to either MIP/ZEO or MIP/5FU cells (FIG. 2C). Thisobservation that MIP/SP cells were more likely to undergo apoptosisfollowing incubation with 5-FU than MIP/ZEO cells was further supportedby significantly higher levels of Caspase 3/7 activity in MIP/SP cellsat 12 hours after incubation with 5-FU than in MIP/ZEO cells(16397.0±2787.6 vs. 9954.0±1104.8, p=0.0003) (FIG. 2D).

In the presence of SPARC, inhibition of Caspase 8 prevents apoptosis inresponse to 5-FU exposure. The relative contribution of the extrinsicpathway in SPARC-mediated apoptosis was examined by assessing the effectof reducing the transcriptional expression of Caspase 8 by siRNA.Effective knock-down of Caspase 8 was achieved by transientlytransfecting MIP/SP or HCT116 cells with 40 nM Caspase 8 siRNA or ascramble oligonucleotide sequence as control. MIP/ZEO and MIP/SP cellswere transiently transfected with Caspase 8 siRNA and cell viability wasassessed following incremental concentrations of 5-FU (0-1200 μM). Itwas noted that knocking-down Caspase 8 gene expression in MIP/ZEO cellsdid not affect their response to 5-FU 1200 μM, as cell viabilitysimilarly decreased from 87.8±5.8 to 52.1±1.7% (p=0.0002) in comparisonto control cells (FIG. 3A). However, Caspase 8 gene silencing in MIP/SPabolished the effect of 5-FU by preventing a decrease in cell viabilityafter treatment with various concentrations of 5-FU (0-1200 μM), incomparison to cells transfected with control scramble siRNA (97.2±1.5%viable untreated cells vs 97.6±1.4% after 5-FU treatment, p=0.8783)(FIG. 3A). In order to further assess whether the effect of Caspase 8gene silencing was dependent on SPARC expression, several CRC cell linesexpressing variable levels of SPARC were examined, such as intrinsicallyhigh SPARC-expressing HCT116 cells and high SPARC-expressing MIP/SPcells; and compared them to low SPARC expressing MIP/ZEO and even lower,MIP/5FU and MIP/CPT cells. Caspase 8 gene knockdown again lessened theeffect of 5-FU on MIP/SP cells, with cell viability in response to 5-FUtreatment increasing by 20.3±4.0% in comparison to cells not transfectedwith Caspase 8 siRNA (p=0.0167) (FIG. 3B). There was no significanteffect of Caspase 8 gene knock-down in MIP/ZEO cells as cell viabilitydecreased following 5-FU treatment despite inhibition of Caspase 8(99.3±1.5% vs. 75.5±0.5%, p=0.0004). Similarly, no effect was seen withCaspase 8 knock-down in MIP/5FU cells, as they remained unaffected by5-FU treatment (FIG. 3B). Using a different chemotherapy, CPT-11, MIP/SPcells had a reduction in cell viability after treatment with CPT-11 100μM (100.0±0.0001% vs. 63.4±4.9%, p=0.00003), which was again abolishedafter transfection with Caspase 8 siRNA despite the presence of CPT-11(98.4±3.1% vs. 97.2±4.0%, p=0.8092) (FIG. 3C). The most interestingfinding was that a similar effect of Caspase 8 gene silencing wasobserved with high SPARC-expressing HCT116 cells, as with MIP/SP cells,with decreased sensitivity to either 5-FU or CPT-11 in comparison tountreated cells (FIGS. 3B and 3C). For example, 88.2±2.3% viable cellsin the presence of Caspase 8 siRNA+5-FU vs. 87.5±5.4% in untreatedcontrols (p=0.88).

The relative contribution of the extrinsic or intrinsic pathways wereexamined in SPARC-mediated apoptosis by assessing the effect of Caspase8 or 9 inhibition on cell viability following in vitro exposure to 5-FU.Using chemical inhibitors that display some specificity against Caspase8-like (z-IETD-fmk) and Caspase 9-like (z-LEHD-fmk•TFA) activities, itwas observed that inhibition of Caspase 8-like activity affected MIP/SPcells more dramatically than control MIP/ZEO cells. In response to 1000μM of 5-FU for 24 hours only, cell viability decreased by 21.0±5.9(p=0.0037) in control MIP/ZEO cells and 16.8±4.5% (p=0.0195) in MIP/SPcells (FIG. 3D). However, in MIP/SP cells, pre-incubation with a Caspase8-like inhibitor prevented a significant decrease in cell viabilityobserved following treatment with 5-FU (FIG. 3D). Cell viability ofMIP/SP cells remained unchanged in the presence or absence of 5-FU,while a decrease in cell viability of 14.0±4.6% (p=0.048) could still beobserved in the 5-FU-treated MIP/ZEO cells. This increase in cellviability in MIP/SP cells despite the presence of 5-FU could be seenfollowing inhibition with as low as 10 μM of Caspase 8-like inhibitor(data not shown), while no such effect could be demonstrated with ahigher concentration of the inhibitor (50 μM) in MIP/ZEO cells.Inhibition of the intrinsic pathway with Caspase 9-like inhibitordesensitized both MIP/ZEO and MIP/SP cells to the effects ofchemotherapy by preventing a significant decrease in cell viability inresponse to 5-FU (FIG. 3D). These results again demonstrate that thereis additional involvement of Caspase 8 in diminishing cell viability inthe presence of higher levels of SPARC.

Given that Caspase 8 gene expression knock-down lessened the effect of5-FU and CPT-11, and enhanced cell viability in MIP/SP cells, it wasnext examined whether this resulted from a reduction in apoptosis.Caspase 3/7 activity was similar in MIP/ZEO cells following incubationwith 5-FU regardless of whether cells were transfected with Caspase 8siRNA (2321.3±661.3) or not (scramble control: 1915.0±661.3; p=0.27).However, in MIP/SP cells, an increase in Caspase 3/7 activity was onlyobserved in control MIP/SP cells (untreated 327.7±30.9 vs. 5-FUtreated5501.0±800.0, p=0.0001), and this effect was abrogated when treatedcells were initially transfected with Caspase 8 siRNA (untreated371.0±58.9 vs. treated 291.0±34.7, p=0.31). No Caspase 3 activation wasobserved in MIP/5FU cells following incubation with 5-FU, eitherfollowing transfection with Caspase 8 siRNA or scramble control (FIG.4A). This reduction in apoptosis was further confirmed by TUNEL assay.In the absence of any Caspase 8 inhibition, the highest percentage ofcells to undergo apoptosis was observed with MIP/SP cells exposed to5-FU, with 20.7±6.9% apoptotic cells, in comparison to the untreatedgroup (5.1±2.0%, p=0.0003). However, following Caspase 8 geneknock-down, there was a decrease in the sensitivity of MIP/SP cells to5-FU (FIG. 4B), with only 0.5±0.5% apoptotic MIP/SP cells detected. Alarge percentage of MIP/ZEO cells continued to undergo apoptosisregardless of the presence or absence of Caspase 8 gene expressionfollowing 5-FU treatment (no transfection with Caspase 8 siRNA:12.2±0.7% apoptotic cells versus transfection with Caspase 8 siRNA:12.5±2.4%, p=0.02). Therefore, in complete contrast to MIP/SP cells,knock-down of Caspase 8 did not inhibit apoptosis in MIP/ZEO cells (FIG.4B, 4C). Resistant MIP/5FU cells did not respond to 5-FU treatmentsignificantly and this was not influenced by changing the expression ofCaspase 8 gene expression (FIG. 4B, 4C).

Based on the above results, there is recruitment of the extrinsicpathway in SPARC-mediated apoptosis, and in particular, this isassociated with a prominent role for Caspase 8. This possibility of adirect SPARC-caspase 8 interaction was examined by assessing bindinginteractions with SPARC by coimmunoprecipitation studies usingantibodies to SPARC, DR4, FADD and Procaspase 8. Different subcellularfractions were examined and only pro-caspase 8 co-immunoprecipitatedwith SPARC in a reciprocal fashion from the cell membrane (FIG. 5A).This interaction between

Procaspase 8 and SPARC disappears when MIP/SP cells were treated with1000 μM 5-FU (FIG. 5A), when co-immunoprecipiation was performed usingan anti-caspase 8 antibody that recognizes the carboxy-terminal sequenceof the p18 fragment of the protein. In the high SPARC-expressing HCT116cells, a similar interaction between pro-caspase 8 and SPARC wasobserved at the cell membrane (FIG. 5B).

Immunofluorescence staining for SPARC and Caspase 8 were also mostprominent in MIP/SP cells and revealed co-localization of SPARC andCaspase 8 expression in the periphery of the cell (FIG. 6A), consistentwith our co-immunoprecipitation results. MIP/ZEO and MIP/5FU cellsshowed minimal SPARC and Caspase 8 expression.

SPARC in combination with 5-FU increases Caspase 8 expression in tumorxenografts. Whether the interaction between SPARC and Caspase 8 couldalso be detected in vivo was next examined. Tumor xenografts bearingMIP/SP cells showed intrinsically higher levels of Caspase 8 geneexpression than MIP/ZEO cells (FIG. 6B). Moreover, Caspase 8 proteinactivity was highest in MIP/SP tumors harvested from animals treatedwith 5-FU (FIG. 6C). We also examined tumors from MIP 101 mousexenografts that had been previously treated with a combination of SPARCand 5-FU, or also as single agents, for Caspase 8 expression, and again,only observed higher levels of Caspase 8 in tumor xenografts harvestedfrom mice that were administered SPARC, and even more significantlyfollowing combination treatment with SPARC and 5-FU (FIG. 6D). Thisup-regulation of Caspase 8 expression in animals exposed to both SPARCand 5-FU appears to be restricted to the tumor xenografts, since liversharvested from mice subjected to this treatment did not have elevatedCaspase 8 expression (FIG. 6D).

Example 6

This Example further localizes the SPARC binding site within theprocapse 8 amino acid sequence.

1. Methods

Cell Lines: MIP101 and HCT116 (ATCC) human CRC cells were maintained inDMEM media supplemented with 1% penicillin-streptomycin, 1% kanamycin(Invitrogen) and 10% newborn calf serum at 37° C. and 5% CO2. For MIP101cells resistant to 5-FU (MIP/5FU) or CPT-11 (MIP/CPT), media were alsosupplemented with 500 μM 5-FU or 10 μM CPT-11, respectively. MIP 101transfected with empty vector (MIP/ZEO) and MIP 101 cells stablytransfected with SPARC (MIP/SP), were also supplemented with 0.01%zeocin (Invitrogen).

Immunoprecipitation: MIP/SP and HCT116 cells were grown until ˜80%confluence, incubated with 1000 μM 5-FU and isolated at 4 hrs. Cellswere separated into nuclear, cytosolic, and membrane fractions usingProteoExtract Subcellular Proteome Extraction Kit (EMD BiosciencesInc.). In addition, in order to verify the site of interaction ofCaspase 8 with SPARC, MIP/SP and MIP/ZEO cells were also incubated withantibodies against Caspase 8 targeting its C-terminus (Cell Signaling)or N-terminus (Abeam) in vitro for 24 hours at 1.5-3.0 μg prior tocollecting and fractionating the cell lysates, for immunoprecipitation,and as well, Caspase 3/7 assay (as described previously). 250 μg of theindividual cellular fractions were incubated with antibodies againstSPARC (10 μg/mL, Haematologic Technologies), Caspase 8 (1:100, CellSignaling Techonology (C-terminus) or Abeam (N-terminus)) or anon-specific anti-mouse IgG antibody as control (Cell SignalingTechnologies), in PBS overnight (4° C.) with gentle agitation.Protein:Antibody mixture was then incubated with 30 μL of Protein A:Protein G (Sigma) (1:1) beads for 4 hrs (4° C.). Proteins were alsoincubated with EZView Red His-Select HC Nickel affinity gel (Sigma) forimmunoprecipitation of His-tagged SPARC protein. For all complexes,beads were washed 5×3 with PBS, eluted with 40 μL of 2×SDS-LoadingBuffer, and used for immunoblotting against SPARC and Caspase 8.

2. Results

The intramolecular localization of the binding interactions betweenProcaspase 8 and SPARC was further studied by co-immunoprecipitationusing antibodies different regions of Procaspase 8. Differentsubcellular fractions were examined by Procaspase 8 co-IP with SPARC ina reciprocal fashion from the MIP/SP (FIG. 7A) and intrinsicallySPARC-overexpressing HCT116 cell membrane fractions (FIG. 7B).Suprisingly, the ability to co-IP of Procaspase 8 and SPARC disappearedwhen MIP/SP cells were exposed to 1000 μM 5-FU (which induces Procaspase8 cleavage) and an anti-caspase 8 antibody that recognizes thecarboxy-terminal sequence of the p18 fragment of the protein was used(FIG. 7A). In contrast, using an anti-caspase 8 antibody recognizing theN-terminal region of this protein, the interaction between SPARC andProcaspase 8 could still be detected despite 5-FU exposure (FIG. 7B).

In order to further validate that the interaction between SPARC andProcaspase 8 occurs within the N-terminus region of Procaspase 8, MIP/SPcells were incubated in vitro with antibodies to Caspase 8 targeting itsN- or C-terminus. Cell lysates subjected to reciprocal co-IP with SPARCor Caspase 8 showed that cells incubated with antibodies targeting theN-terminus of Caspase 8 no longer interacted with SPARC, whileincubation with antibodies targeting the C-terminus had no such effect(FIG. 7C). By interfering with this interaction, MIP/SP cells becameless responsive to 5-FU as indicated by a significant reduction inapoptosis (FIG. 7D).

Thus, this Example demonstrates an interaction between SPARC andProcaspase 8, taking place at the amino p43/p41 fragment of Caspase 8containing the N-terminal DED-domains and the catalytic site (FIG. 7E).

Example 7

This example demonstrates that Procaspase 8 binds to a SPARC domainwhich is amino (upstream) of the SPARC follistatin-like domain domain.

Immunopreciptation was performed with anti-histidine tag antibody (IP:His) (See FIG. 8A) or an anti-Caspase 8 antibody (IP: Capsaase 8) (SeeFIG. 8B) in the presence of Procaspase 8 and truncated SPARCpolypeptides comprising a sequence near the SPARC amino terminus (SEQ IDNO: 20) (MIP/NT), a sequence from the SPARC follistatin-like domain (SEQID NO: 22) (MIP/FS), a sequence from the SPARC extracellular domain (SEQID NO: 24) (MIP/EC) or a control antibody (IgG). These studies confirmthat the interaction between SPARC and Procaspase 8 is specific andinvolves a SPARC domain which is within the amino acid sequence of SEQID NO: 20.

Example 8

This example demonstrates that Bcl-2 interacts with Procaspase 8 andthat the site of interaction between Bcl-2 and Procaspase 8 is in thesame region where SPARC interacts with Procaspase 8. This examplefurther demonstrates that loss of Bcl-2 binding to Procaspase 8 by SPARCresults in increased sensitivity of cells to chemotherapy.

The cell lines MIP101, RKO, MiaPaca, and MCF-7 cells were maintained inDMEM media supplemented with 1% penicillin-streptomycin, 1% kanamycin(Invitrogen) and 10% newborn calf serum at 37° C. and 5% CO₂. For MIP101cells resistant to 5-FU (MIP/5FU) or CPT-11 (MIP/CPT), media were alsosupplemented with 500 μM 5-FU or 10 μM CPT-11, respectively. For RKOcells resistant to 5-FU (RKO/5FU), or CPT-11 (RKO/CPT), media were alsosupplemented with 25 μM 5-FU, or 18 μM CPT-11, respectively. For MiaPacacells resistant to CPT, media was also supplemented with 100 μM CPT-11.For MCF-7 cells resistant to cisplatin, media was supplemented with 30μM cisplatin. MIP101 transfected with empty vector (MIP/ZEO) or MIP 101cells stably transfected with SPARC (MIP/SP), were also supplementedwith 0.01% zeocin (Invitrogen).

Cells were grown until ˜80% confluence, and lysates were separated intonuclear, cytosolic, and membrane fractions. 250 μg of individualcellular fractions were incubated with antibodies against HIS (1:100,Sigma), capsase 8 (N-terminus) (1:100, Abcam), Bcl-2 (1:100, CellSignaling Technologies) or a non-specific anti-mouse IgG antibody ascontrol (Cell Signaling Technologies), in PBS overnight (4° C.).Protein:Antibody mixture was then incubated with 30 mL of ProteinA:Protein G (Sigma) (1:1) beads for 4 hours (4° C.). For all complexes,beads were washed with PBS, and eluted with 2×SDS-loading buffer, andused for immunoblotting against caspase 8, HIS, or Bcl-2.

In order to determine the region of interaction between Bcl-2 andProcaspase 8, cells were also incubated with antibodies against caspase8 targeting its C-terminus (Cell Signaling) or N-terminus (Abcam) invitro for 24 hours at 1.5-3.0□g prior to collecting and fractionatingthe cell lysates for immunoprecipitation. In addition, to assess ifexogenous SPARC interferes with the interaction of procaspase 8 andBcl-2, cells were incubated with 10 □g/ml of SPARC for 72-hrs, in thepresence or absence of antibodies against SPARC (3 □g, HematologicTechnologies) for 24 hrs, prior to collection for cellular fractionationand immunoprecipitation.

In resistant cell lines (MIP101 colorectal cancer cells resistant to5-FU, MIP/5FU; pancreatic cancer cells resistant to CPT, MiaPaca/CPT),these studies demonstrate that Bcl-2 interacts with Procaspase 8 (FIGS.9A and 9C). It is further demonstrated that this interaction betweenBcl-2 and Procaspase 8 can be blocked by the use of antibodies directedtoward the N-terminus of Procapase 8 (FIG. 9B) and as well, with theaddition of exogenous SPARC (FIG. 9C). It is further demonstrated thatinterference of Bcl-2 binding to Procaspase 8 by SPARC results inincreased cellular chemotherapeutic sensitivity.

Example 9

This example demonstrates that the interaction between Bcl-2 andProcaspase 8 occurs in the region of the DEDI and DEDII domains ofProcaspase 8.

Site-directed mutagenesis was carried out as follows: Procaspase 8 cDNAwas cloned into pcDNA3.1/myc-His. 100 ng of plasmid DNA was methylatedwith DNA methylase (4 U/μL) and 10×SAM's buffer as per manufacturer'sprotocol (Gene Tailor Site-Directed Mutagenesis system, Invitrogen) for1 hour at 37° C. Methylated DNA was then subjected to mutagenesis, usingthe following specific primers to target mutations within the DEDIdomain (forward 5′ tctttagaaa tactataacc AGGTACCATC AGGTACCGTGtagaccggag 3′, reverse 5′ ggttatagta tttctaaaga cgacttcagg 3′); putativebinding (PB) region (forward 5′ ttgac ctgtcacttc tagaAATAGC ggagttcaag3′, reverse 5′ tctagaagtgacag gtcaacaagg ggtt 3′); and DEDII domain(forward 5′ gagatagtct aaagtcttct CCTAGTACAG GAGCTAACTC ccagaaaatt 3′,reverse 5′ agaagacttt agctatctc gtactgggac 3′). All mutations wereverified by DNA sequencing. Once verified, vectors expressing either thewild type procaspase 8 (Csp 8), or the mutant procaspase 8 withmutations in the DEDI domain (DEDI), protein binding domain (PB) orDEDII domain (DEDII) were then used for transient transfection intocells.

The cell lines and cell lysate procedures described in Example 8 wereused in Example 9. The site-directed mutagenesis studies demonstratedthat mutations to the DEI and DEDII domains of Procaspase 8 abolishedthe ability of Procaspase 8 to bind with Bcl-2 (FIG. 10A). Inparticular, as demonstrated in FIG. 10A, transient transfection ofMIP/5FU and MiaPaCa/CPT with vectores containing Procaspase 8 withmutations wither within the DEDI or DEDII domains failed toimmunoprecipitate Bcl-2 in a reciprocal fashion as compared with cellstransfected with wild-type Procaspase 8. A potential model for theinteraction between Procaspase 8, SPARC, and Bcl-2 is provided in FIG.10B.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

I claim:
 1. A pharmaceutical composition, the composition comprising atherapeutic or diagnostic agent coupled to a Procaspase 8 polypeptideand a pharmaceutically acceptable carrier, wherein the therapeutic ordiagnostic agent is for the treatment or diagnosis of a cancer.
 2. Thepharmaceutical composition of claim 1, wherein the Procaspase 8polypeptide comprises an amino acid sequence of any one of SEQ ID NOS:1-18, 25, and
 26. 3. The pharmaceutical composition of claim 1, whereinthe cancer is a tumor.
 4. The pharmaceutical composition of claim 1,wherein the diagnostic agent is selected from the group consisting ofradioactive agents, MRI contrast agents, X-ray contrast agents,ultrasound contrast agents, and PET contrast agents.
 5. Thepharmaceutical composition of claim 1, wherein the therapeutic agent isselected from the group consisting of docetaxel, paclitaxel, taxanes,platinum compounds, antifolates, antimetabolites, antimitotics, DNAdamaging agents, proapoptotics, differentiation inducing agents,antiangiogenic agents, antibiotics, hormones, peptides, antibodies,tyrosine kinase inhibitors, kinase inhibitors, anti-vascular endothelialgrowth factor compounds (anti-VEGFs), anti-epidermal growth factorreceptor compounds (anti-EGFRs), tTF, TNF, radionuclides, andcombinations thereof.
 6. The pharmaceutical composition of claim 1,wherein the therapeutic agent is selected from the group consisting ofgenistein, adriamycin, ansamycin, asparaginase, bleomycin, busulphan,cisplatin, carboplatin, carmustine, capecitabine, chlorambucil,cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin,daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide,epothilones, floxuridine, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine,mechlorethamine, mercaptopurine, melphalan, methotrexate, rapamycin(sirolimus), mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel,pamidronate, pentostatin, plicamycin, procarbazine, rituximab,streptozocin, teniposide, thioguanine, thiotepa, taxanes, vinblastine,vincristine, vinorelbine, taxol, combretastatins, discodermolides,transplatinum, 5-fluorouracil, radionuclides, and combinations thereof.7. The pharmaceutical composition of claim 5, wherein the therapeuticagent comprises particles of paclitaxel and wherein more than 50% of thetherapeutic agent is in nanoparticle form.
 8. The pharmaceuticalcomposition of claim 5, wherein the therapeutic agent is an antibody orantibody fragment that mediates one or more of complement activation,cell mediated cytotoxicity, and opsinization.
 9. A method for deliveringa therapeutic or diagnostic agent to a disease site in a mammal, whereinthe disease is cancer, and wherein said method comprises administeringto the mammal a therapeutically or diagnostically effective amount of apharmaceutical composition of claim
 1. 10. The method of claim 9,wherein the Procaspase 8 polypeptide comprises an amino acid sequence ofany one of SEQ ID NOS: 1-18, 25, and
 26. 11. The method of claim 9,wherein the mammal is a human.
 12. The method of claim 9, wherein thedisease site is a tumor.
 13. The method of claim 12, wherein the tumoris located in the lung, prostate, head, neck, bladder, liver, ovary,kidney, gut, brain, or breast.
 14. The method of claim 9, wherein theroute of administration is selected from the group consisting ofintravenous, subcutaneous, intramuscular, intraperitoneal, intratumoral,oral, and inhalational.
 15. The method of claim 9, wherein thediagnostic agent is selected from the group consisting of radioactiveagents, MRI contrast agents, X-ray contrast agents, ultrasound contrastagents, and PET contrast agents.
 16. The method of claim 9, wherein thetherapeutic agent is selected from the group consisting of docetaxel,paclitaxel, taxanes, platinum compounds, antifolates, antimetabolites,antimitotics, DNA damaging agents, proapoptotics, differentiationinducing agents, antiangiogenic agents, antibiotics, hormones, peptides,antibodies, tyrosine kinase inhibitors, kinase inhibitors, anti-vascularendothelial growth factor compounds (anti-VEGFs), anti-epidermal growthfactor receptor compounds (anti-EGFRs), tTF, TNF, radionuclides, andcombinations thereof.
 17. The method of claim 16, wherein thetherapeutic agent is selected from the group consisting of genistein,adriamycin, ansamycin, asparaginase, bleomycin, busulphan, cisplatin,carboplatin, carmustine, capecitabine, chlorambucil, cytarabine,cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin,dexrazoxane, docetaxel, doxorubicin, etoposide, epothilones,floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea,idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine,mercaptopurine, melphalan, methotrexate, rapamycin (sirolimus),mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate,pentostatin, plicamycin, procarbazine, rituximab, streptozocin,teniposide, thioguanine, thiotepa, taxanes, vinblastine, vincristine,vinorelbine, taxol, combretastatins, discodermolides, transplatinum,5-fluorouracil, radionuclides, and combinations thereof.
 18. The methodof claim 16, wherein the therapeutic agent comprises particles ofpaclitaxel and wherein more than 50% of the therapeutic agent is innanoparticle form.
 19. The method of claim 16, wherein the therapeuticagent is an antibody or antibody fragment that mediates one or more ofcomplement activation, cell mediated cytotoxicity, and opsinization. 20.The method of claim 12, wherein the tumor is selected from oral cavitytumors, pharyngeal tumors, digestive system tumors, the respiratorysystem tumors, bone tumors, cartilaginous tumors, bone metastases,sarcomas, skin tumors, melanoma, breast tumors, the genital systemtumors, urinary tract tumors, orbital tumors, brain and central nervoussystem tumors, gliomas, endocrine system tumors, thyroid tumors,esophageal tumors, gastric tumors, small intestinal tumors, colonictumors, rectal tumors, anal tumors, liver tumors, gall bladder tumors,pancreatic tumors, laryngeal tumors, tumors of the lung, bronchialtumors, non-small cell lung carcinoma, small cell lung carcinoma,uterine cervical tumors, uterine corpus tumors, ovarian tumors, vulvartumors, vaginal tumors, prostate tumors, prostatic carcinoma, testiculartumors, tumors of the penis, urinary bladder tumors, tumors of thekidney, tumors of the renal pelvis, tumors of the ureter, head and necktumors, parathyroid cancer, Hodgkin's disease, Non-Hodgkin's lymphoma,multiple myeloma, leukemia, acute lymphocytic leukemia, chroniclymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia,and a combination thereof.
 21. The method of claim 12, wherein the tumoris a sarcoma, adenocarcinoma, squamous cell carcinoma, large cellcarcinoma, small cell carcinoma, basal cell carcinoma, clear cellcarcinoma, oncytoma, or a combination thereof.
 22. The method of claim9, wherein the cancer is colorectal cancer.
 23. The method of claim 9,wherein the cancer is breast cancer.
 24. The method of claim 9, whereinthe cancer is head and neck cancer.
 25. The method of claim 9, whereinthe cancer is non-small cell lung cancer.
 26. The method of claim 9,wherein the cancer is pancreatic cancer.
 27. The method of claim 9,wherein the cancer is melanoma.
 28. The method of claim 9, wherein thecancer is bladder cancer.
 29. A kit for diagnosing or treating a diseasein a mammal wherein said kit comprises a pharmaceutical composition ofclaim 1; and instructions for use in the diagnosis and/or treatment ofcancer.
 30. The kit of claim 29, wherein the Procaspase 8 polypeptidecomprises an amino acid sequence of any one of SEQ ID NOS: 1-18, 25, and26.